Genome-Wide Association Study of African and European Americans Implicates Multiple Shared and Ethnic Specific Loci in Sarcoidosis Susceptibility

<div><p>Sarcoidosis is a systemic inflammatory disease characterized by the formation of granulomas in affected organs. Genome-wide association studies (GWASs) of this disease have been conducted only in European population. We present the first sarcoidosis GWAS in African Americans (AAs, 818 cases and 1,088 related controls) followed by replication in independent sets of AAs (455 cases and 557 controls) and European Americans (EAs, 442 cases and 2,284 controls). We evaluated >6 million SNPs either genotyped using the Illumina Omni1-Quad array or imputed from the 1000 Genomes Project data. We identified a novel sarcoidosis-associated locus, <em>NOTCH4</em>, that reached genome-wide significance in the combined AA samples (rs715299, <em>P</em>_AA-meta = 6.51×10⁻¹⁰) and demonstrated the independence of this locus from others in the MHC region in the same sample. We replicated previous European GWAS associations within <em>HLA-DRA, HLA-DRB5, HLA-DRB1</em>, <em>BTNL2,</em> and <em>ANXA11</em> in both our AA and EA datasets. We also confirmed significant associations to the previously reported <em>HLA-C</em> and <em>HLA-B</em> regions in the EA but not AA samples. We further identified suggestive associations with several other genes previously reported in lung or inflammatory diseases.</p> </div

Sample summary before and after quality control (QC).

a<p>Taken from the Illumina YRI-ASW iControlDB;</p>b<p>175 Caucasian healthy controls from the Illumina iControlDB, 1047 controls from the dbGaP GENEVA Melanoma study, and 1986 controls from the dbGAP CIDR: NGRC Parkinson’s Disease Study.</p

Regions of association meeting genome-wide significance and their most significant SNPs grouped by sample.

1<p>Major/minor allele of AAs as the reference;</p>2<p>Minor allele frequency;</p>3<p>The odds ratio (OR) was calculated with respect to the minor allele of AAs.</p>a<p>Previously reported sarcoidosis loci meeting genome-wide significance in the AA discovery set.</p>b<p>Potentially novel region meeting genome-wide significance after the meta-analysis of AA datasets.</p>c<p>Previously reported sarcoidosis loci meeting genome-wide significance in the EA dataset.</p><p>Note that stepwise conditional analysis results to identify independent signals within the MHC region can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043907#pone.0043907.s006" target="_blank">Tables S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043907#pone.0043907.s007" target="_blank">S4</a>.</p

Replication of previously reported SNPs associated with sarcoidosis [9], [11], [25], [27].

1<p>Major/minor allele of AAs as the reference;</p>2<p>Minor allele frequency;</p>3<p>The odds ratio (OR) was calculated with respect to the minor allele of AAs.</p

Path analysis model used to estimate the effect of smoking duration on SS and the role of potential confounding factors.

<p>The standardized path coefficients, significant odds ratios (OR) and p-values (in bold) are shown next to each path. A. Effect of smoking duration on SS, conditional on socioeconomic status; B. Model used to estimate the effect of smoking duration on SS, conditional on body mass index.</p

Logistic Regression models exploring the effect of covariates of smoking on the risk of classification as Sjögren’s Syndrome.

<p>The only significant predictor was smoking duration.</p

Functional characterizations of <i>OAS1</i> isoforms.

<p>(A) Protein expression of OAS1 isoforms was evaluated in EBV-transformed B cells from SS patients (four independent samples from each genotype group) using anti-OAS1 antibody targeting the shared epitope of all the isoforms. The stimulated cells were treated with universal type I IFN (1500U/ml) for 24hrs. The p44 isoform was not detectable using western-blot due to its low expression. The right panel shows quantified band intensity normalized to the GAPDH in each sample. (B) The transcript levels of each <i>OAS1</i> isoform from the same sets of cells described above were determined using real-time PCR. Consistent with the RNA-seq results, the SS-associated risk allele A of rs10774671 was correlated with decreased levels of p46 and increased expression of the p42, p48, and p44 isoforms (significance levels are shown at the bottom). The transcript levels of all the isoforms significantly increased after IFN stimulation (two-tailed <i>t</i> test); however, only p46 had increased protein production after IFN stimulation. (Significance level: ** <i>P</i><0.01; *** <i>P</i><0.001) (C) Individual isoforms of <i>OAS1</i> tagged with Xpress epitope were cloned and transfected into HEK 293T cells for 48hrs. The p48 and p44 isoforms had impaired protein expression compared to p46 and p42, although their transcript levels were equivalent as determined by real-time PCR (n = 4; normalized to <i>HMBS</i>). (D) The full-length and truncated <i>OAS1</i> p48 and p44 isoforms were cloned into HEK 293T cells. Western-blot indicated the lack of expression of the full-length p48 and p44 isoforms, whereas the truncation of both isoform transcripts (T2 and T4) was able to restore protein expression. (E) The 3' alternatively spliced terminus of different <i>OAS1</i> isoforms were linked to the 3'-end of GFP to observe their influence on GFP protein expression in HEK 293T cells. The 3'-terminus from the p48 and p44 isoforms resulted in decreased expression of GFP.</p

Differentially expressed transcripts between 115 anti-Ro/SSA positive SS cases and 56 controls identified through transcriptome profiling.

<p>(A) We identified 73 genes (represented by 83 probes on the heatmap) differentially expressed between anti-Ro/SSA positive SS cases and healthy controls (absolute FC >2 and <i>q</i><0.05). Among the differentially expressed genes, 57 were type I IFN-regulated genes (black bar on right) and formed an IFN signature where most genes were overexpressed in SS patients (yellow indicates overexpressed genes compared to controls). (B) The 57 differentially expressed type I IFN-regulated genes were re-clustered in anti-Ro/SSA positive SS cases using <i>k</i>-means (<i>k</i> = 3) algorithm and heterogeneity of the IFN signature levels in anti-Ro/SSA positive SS cases was observed.</p

Study design.

<p>To evaluate genetic factors involved in the dysregulation of type I IFN signaling in SS, we first compared transcriptional profiles between anti-Ro/SSA positive SS cases and controls to identify genes that make up the IFN signature in SS. We then performed genetic association analysis for variants in the regions of the differentially expressed genes. By integrating transcriptome data with genotype data, <i>cis</i>-eQTL analysis was performed for SS-associated SNPs to evaluate their role in gene dysregulation. This genomic convergence approach resulted in increased power to identify and prioritize disease susceptibility genes for further genetic replication and functional studies.</p

Composition of independent cohorts used in the genetic association analyses.

<p>Composition of independent cohorts used in the genetic association analyses.</p